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Cyclic Gmp-Binding, Cyclic Gmp-Specific Europäisches Patentamt *EP000652960B9* (19) European Patent Office Office européen des brevets (11) EP 0 652 960 B9 (12) CORRECTED EUROPEAN PATENT SPECIFICATION Note: Bibliography reflects the latest situation (15) Correction information: (51) Int Cl.7: C12N 15/55, C12N 9/16, Corrected version no 1 (W1 B1) C07K 16/40, C07K 16/46, Corrections, see page(s) 5, 49-51 C12N 15/11 (48) Corrigendum issued on: (86) International application number: 27.10.2004 Bulletin 2004/44 PCT/US1994/006066 (45) Date of publication and mention (87) International publication number: of the grant of the patent: WO 1994/028144 (08.12.1994 Gazette 1994/27) 07.03.2001 Bulletin 2001/10 (21) Application number: 94919288.4 (22) Date of filing: 27.05.1994 (54) CYCLIC GMP-BINDING, CYCLIC GMP-SPECIFIC PHOSPHODIESTERASE MATERIALS AND METHODS CYCLISCH GMP BINDENDE CYCLISCH GMP SPEZIFISCHE PHOSPHODIESTERASE MATERIALIEN UND VERFAHREN. PHOSPHODIESTERASE DE FIXATION DU MONOPHOSPHATE DE GUANOSINE CYCLIQUE ET AVEC SPECIFICITE POUR CELUI-CI ET PROCEDE D’OBTENTION (84) Designated Contracting States: • FERGUSON, Kenneth, M. AT BE CH DE DK ES FR GB GR IE IT LI LU MC NL Bothell, WA 98021 (US) PT SE • FRANCIS, Sharron, H. Nashville, TN 37215 (US) (30) Priority: 27.05.1993 US 68051 • KADLECEK, Ann Seattle, WA 98103 (US) (43) Date of publication of application: • LOUGHNEY, Kate 17.05.1995 Bulletin 1995/20 Seattle, WA 98115 (US) • McALLISTER-LUCAS, Linda, M. (83) Declaration under Rule 28(4) EPC (expert Nashville, TN 37212 (US) solution) • SONNENBURG, William, K. Mountlake Terrace, WA 98043 (US) (60) Divisional application: • THOMAS, Melissa, K. 00112074.0 / 1 038 963 Boston, MA 02114 (US) (73) Proprietors: (74) Representative: Richardson, Kate et al • BOARD OF REGENTS Forrester & Boehmert, OF THE UNIVERSITY OF WASHINGTON Pettenkoferstrasse 20-22 Seattle, WA 98105 (US) 80336 München (DE) • ICOS CORPORATION Bothell, WA 98021 (US) (72) Inventors: • BEAVO, Joseph, A. Seattle, WA 98155 (US) • CORBIN, Jackie, D. Nashville, TN 37215 (US) Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 0 652 960 B9 Printed by Jouve, 75001 PARIS (FR) (Cont. next page) EP 0 652 960 B9 (56) References cited: • JOURNAL OF BIOLOGICAL CHEMISTRY., • JOURNAL OF BIOLOGICAL CHEMISTRY., vol.266, no.26, 15 September 1991, BALTIMORE vol.265, no.25, 5 September 1990, BALTIMORE US pages 17655 - 17661 SONNENBURG ET AL. US pages 14971 - 14978 THOMAS ETAL. ’Molecular Cloning of a Cyclic GMP-stimulated ’Substrate- and Kinase-directed regulation of Cyclic Nucleotide Phosphodiesterase cDNA’ Phosphorylation of a cGMP-binding • GENOMICS, vol.13, no.3, July 1992 pages 698 - Phosphodiesterase by cGMP.’ cited in the 704 COLLINS ET AL. ’The human Beta-Subunit application of Rod Photoreceptor cGMP • JOURNAL OF BIOLOGICAL CHEMISTRY., Phosphodiesterase: Complete Retinal cDNA vol.265, no.25, 5 September 1990, BALTIMORE, Sequence and Evidence for Expression in Brain’ MD US pages 14964 - 14970 THOMAS ET AL. cited in the application ’Characterization of a Purified Bovine Lung • JOURNAL OF BIOLOGICAL CHEMISTRY., cGMP-binding cGMP Phosphodiesterase’ cited vol.268, no.30, 25 October 1993, BALTIMORE US in the application pages 22863 - 22873 MCALLISTER-LUCAS ET • JOURNAL OF BIOLOGICAL CHEMISTRY., AL. ’The Structure of a Bovine Lung vol.267, no.26, 15 September 1992, BALTIMORE cGMP-binding, cGMP-specific US pages 18683 - 18688 REPASKE ET AL. ’A Phosphodiesterase Deduced from a cDNA Polymerase Chain Reaction Strategy to Identify Clone’ and Clone Cylic Nucleotide Phosphodiesterase cDNAs’ 2 EP 0 652 960 B9 (W1B1) Description [0001] Experimental work described herein was supported in part by Research Grants GM15731, DK21723, DK40029 and GM41269 and the Medical Scientist Training Program Grant GM07347 awarded by the National Institutes 5 of Health. The United States government has certain rights in the invention. FIELD OF THE INVENTION [0002] The present invention relates generally to a cyclic guanosine monophosphate-binding, cyclic guanosine 10 monophosphate-specific phosphodiesterase designated cGB-PDE and more particularly to novel purified and isolated polynucleotides encoding cGB-PDE polypeptides, to methods and materials for recombinant production of cGB-PDE polypeptides, and to methods for identifying modulators of cGB-PDE activity. BACKGROUND 15 [0003] Cyclic nucleotide phosphodiesterases (PDEs) that catalyze the hydrolysis of 3'5' cyclic nucleotides such as cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) to the corresponding nucle- oside 5' monophosphates constitute a complex family of enzymes. By mediating the intracellular concentration of the cyclic nucleotides, the PDE isoenzymes function in signal transduction pathways involving cyclic nucleotide second 20 messengers. [0004] A variety of PDEs have been isolated from different tissue sources and many of the PDEs characterized to date exhibit differences in biological properties including physicochemical properties, substrate specificity, sensitivity to inhibitors, immunological reactivity and mode of regulation. [See Beavo et al., Cyclic Nucleotide Phosphodiesterases: Structure, Regulation and Drug Action, John Wiley & Sons, Chichester, U.K. (1990)] Comparison of the known amino 25 acid sequences of various PDEs indicates that most PDEs are chimeric multidomain proteins that have distinct catalytic and regulatory domains. [See Charbonneau, pp. 267-296 in Beavo et al., supra] All mammalian PDEs characterized to date share a sequence of approximately 250 amino acid residues in length that appears to comprise the catalytic site and is located in the carboxyl terminal region of the enzyme. PDE domains that interact with allosteric or regulatory molecules are thought to be located within the amino-terminal regions of the isoenzymes. Based on their biological 30 properties, the PDEs may be classified into six general families: the Ca2+/calmodulin-stimulated PDEs (Type I), the cGMP-stimulated PDEs (Type II), the cGMP-inhibited PDEs (Type III), the cAMP-specfic PDEs (Type IV), the cGMP- specific phosphodiesterase cGB-PDE (Type V) which is the subject of the present invention and the cGMP-specific photoreceptor PDEs (Type VI). [0005] The cGMP-binding PDEs (Type II, Type V and Type VI PDEs), in addition to having a homologous catalytic 35 domain near their carboxyl terminus, have a second conserved sequence which is located closer to their amino terminus and which may comprise an allosteric cGMP-binding domain. See Charbonneau et al., Proc. Natl. Acad. Sci. USA, 87: 288-292 (1990). [0006] The Type II cGMP-stimulated PDEs (cGs-PDEs) are widely distributed in different tissue types and are thought to exist as homodimers of 100-105 kDa subunits. The cGs-PDEs respond under physiological conditions to elevated 40 cGMP concentrations by increasing the rate of cAMP hydrolysis. The amino acid sequence of a bovine heart cGs-PDE and a partial cDNA sequence of a bovine adrenal cortex cGs-PDE are reported in LeTrong et al., Biochemistry, 29: 10280-10288 (1990) and full length bovine adrenal and human fetal brain cGs-PDE cDNA sequences are described in Patent Cooperation Treaty International Publication No. WO 92/18541 published on October 29, 1992. The full length bovine adrenal cDNA sequence is also described in Sonnenburg et al., J. Biol. Chem., 266: 17655-17661 (1991). 45 [0007] The photoreceptor PDEs and the cGB-PDE have been described as cGMP-specific PDEs because they ex- hibit a 50-fold or greater selectivity for hydrolyzing cGMP over cAMP. [0008] The photoreceptor PDEs are the rod outer segment PDE (ROS-PDE) and the cone PDE (COS-PDE). The holoenzyme structure of the ROS-PDE consists of two large subunits α (88 kDa) and β (84 kDa) which are both cat- alytically active and two smaller γ regulatory subunits (both 11 kDa). A soluble form of the ROS-PDE has also been 50 identified which includes α, β, and γ subunits and a δ subunit (15 kDa) that appears to be identical to the COS-PDE 15 kDa subunit. A full-length cDNA corresponding to the bovine membrane-associated ROS-PDE α subunit is described in Ovchinnikov et al., FEBS Lett., 223: 169-173 (1987) and a full length cDNA corresponding to the bovine rod outer segment PDE β subunit is described in Lipkin et al., J. Biol. Chem., 265: 12955-12959 (1990). Ovchinnikov et al., FEBS Lett., 204: 169-173 (1986) presents a full-length cDNA corresponding to the bovine ROS-PDE γ subunit and the amino 55 acid sequence of the δ subunit. Expression of the ROS-PDE has also been reported in brain in Collins et al., Genomics, 13: 698-704 (1992). The COS-PDE is composed of two identical α' (94 kDa) subunits and three smaller subunits of 11 kDa, 13 kDa and 15 kDa. A full-length cDNA corresponding to the bovine COS-PDE α' subunit is reported in Li et al., Proc. Natl. Acad. Sci. USA, 87: 293-297 (1990). 3 EP 0 652 960 B9 (W1B1) [0009] cGB-PDE has been purified to homogeneity from rat [Francis et al., Methods Enzymol., 159: 722-729 (1988)] and bovine lung tissue [Thomas et al., J. Biol. Chem., 265: 14964-14970 (1990), hereinafter "Thomas I"]. The presence of this or similar enzymes has been reported in a variety of tissues and species including rat and human platelets [Hamet et al., Adv. Cyclic Nucleotide Protein Phosphorylation Res., 16: 119-136 (1984)], rat spleen [Coquil et al., 5 Biochem. Biophys. Res. Commun., 127: 226-231 (1985)], guinea pig lung [Davis et al., J. Biol. Chem., 252: 4078-4084 (1977)], vascular smooth muscle [Coquil et al., Biochim. Biophys. Acta, 631: 148-165 (1980)], and sea urchin sperm [Francis et al., J. Biol. Chem., 255: 620-626 (1979)].
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